Both the emergence and subsequent human-to-human transmission of SARS-CoV in 2002-2003, and ofthe highly virulent human coronavirus HCoV-EMC in the Middle East and Europe in 2012-2013 exemplifies CoV movement potential and transmissibility, and underscores the urgent and critical need for a broadly efficacious therapeutics. The overall goal of Project 2 is to identify inhibitors of two highly conserved CoV processes, replication fidelity and RNA capping, that are essential for SARS-CoV virulence and survival in vivo. Multiple viral proteins and enzymatic activities are critical for these processes, including CoV 3'-to-5' exoribonuclease (fidelity; nsp14-ExoN) and 2'-0-methyltransferase (capping; nsp16-0MTase) activities. Consistent with the importance of these processes, we have shown that decreased replication fidelity and ablation of RNA capping through genetic inactivation of either ExoN or OMTase, respectively, results in replication competent viruses that are profoundly attenuated in vivo. In Aims 1 and 2, we will work with the Screening Core (Core B) and the Medicinal Chemistry lead Development Core (Core C) to identify, characterize, and optimize small molecule inhibitors of SARS-CoV fidelity and RNA capping. Once active compounds are identified, we will define their mechanism of action, test for the development of virus resistance, and determine their activity across the CoV family. In Aim S, we will work with Core C to chemically optimize and test the in vivo efficacy of lead compounds in progressively tiered models of SARSCoV disease severity, and assess the development of drug resistance in vivo. The complementary expertise ofthe Denison and Baric Labs, extensive preliminary datasets, state-of-the-art technologies, and the expertise of SR in the areas of medicinal chemistry, high-throughput screening, and drug development will contribute significantly to the successful identification, confirmation, and in vivo testing of lead compounds. Ultimately, inhibiting these two conserved and distinct pathways required for in vivo pathogenesis will allow for the treatment of endemic and emerging CoVs and potentially reduce the emergence of viral resistance.